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Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors.

Peron M, Lovisa F, Poli E, Basso G, Bonvini P - PLoS ONE (2015)

Bottom Line: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation.We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity.However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity.

View Article: PubMed Central - PubMed

Affiliation: Clinica di Oncoematologia Pediatrica di Padova, Azienda Ospedaliera-Università di Padova, Padua, Italy.

ABSTRACT

Background: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation. This concept is essential from a therapeutic standpoint, as clinical evidence indicates that tumours carrying deregulated RTKs are particularly susceptible to their activity but also to their inhibition. Rhabdomyosarcoma (RMS) is an aggressive childhood cancer where emerging therapies rely on the use kinase inhibitors, and among druggable kinases ALK represents a potential therapeutic target to commit efforts against. However, the functional relevance of ALK in RMS is not known, likewise the multi-component deregulated RTK profile to which ALK belongs.

Methods: In this study we used RMS cell lines representative of the alveolar and embrional histotype and looked at ALK intracellular localization, activity and cell signalling.

Results: We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity. Indeed, increase of ALK phosphorylation was observed upon PTPase inhibition, as well as after ligand binding or protein overexpression. In these conditions, ALK signalling proceeded through the MAPK/ERK and PI3K/AKT pathways, and it was susceptible to ATP-competitive inhibitors exposure. However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity. Indeed, analysis of baseline and inducible RTK phosphorylation confirmed that RMS cells were susceptible to ALK kinase inhibitors even in the absence of the primary intended target, due to the presence of compensatory RTKs signalling pathways.

Conclusions: These data, hence, provided evidences of a potentially active role of ALK in RMS cells, but also suggest caution in considering ALK a major therapeutic target in this malignancy, particularly if expression and activity cannot be accurately determined.

No MeSH data available.


Related in: MedlinePlus

Endogenous ALK expression levels influence receptor intrinsic kinase activity in RMS cells.(A) ALK protein expression (upper panels) and phosphorylation (lower panel) in RMS (RH30, RH4, RD) and neuroblastoma (NB1, SH-SY5Y) cell lines. Western blot analysis of cell extracts using the specified polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved ALK form (140 kDa) are shown. ALK gene status is also indicated (A = amplified; M = mutated; WT = wild-type) (B) Cell surface detection and quantitative assessment of ALK receptor. Membrane-bound ALK kinase in non-permeabilized RMS and neuroblastoma cells using a primary monoclonal antibody against the N-terminal portion of ALK. Fluorescent-conjugated secondary antibody was used to acquire ALK signal with FACS-Calibur Cell Cytometer. ALK-amplified NB1 cells were included in the analysis as positive control for ALK expression and localization at the plasma membrane. (C) mAb46 effect on ALK receptor activation. RMS (RH30, RH4 and RD) and neuroblastoma (NB1, SHSY-5Y) cells were treated (+) or left untreated (-) with 1 μg/ml agonist mAb46 for 30 min. Membrane-bound (MB) and intracellular (IC) ALK was immunoprecipitated as described in material and methods and detected using polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved (140 kDa) ALK proteins position is indicated by arrowheads. NB1 cell extracts were used as positive control for ALK expression in RH4 and RD immunoblots.
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pone.0132330.g001: Endogenous ALK expression levels influence receptor intrinsic kinase activity in RMS cells.(A) ALK protein expression (upper panels) and phosphorylation (lower panel) in RMS (RH30, RH4, RD) and neuroblastoma (NB1, SH-SY5Y) cell lines. Western blot analysis of cell extracts using the specified polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved ALK form (140 kDa) are shown. ALK gene status is also indicated (A = amplified; M = mutated; WT = wild-type) (B) Cell surface detection and quantitative assessment of ALK receptor. Membrane-bound ALK kinase in non-permeabilized RMS and neuroblastoma cells using a primary monoclonal antibody against the N-terminal portion of ALK. Fluorescent-conjugated secondary antibody was used to acquire ALK signal with FACS-Calibur Cell Cytometer. ALK-amplified NB1 cells were included in the analysis as positive control for ALK expression and localization at the plasma membrane. (C) mAb46 effect on ALK receptor activation. RMS (RH30, RH4 and RD) and neuroblastoma (NB1, SHSY-5Y) cells were treated (+) or left untreated (-) with 1 μg/ml agonist mAb46 for 30 min. Membrane-bound (MB) and intracellular (IC) ALK was immunoprecipitated as described in material and methods and detected using polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved (140 kDa) ALK proteins position is indicated by arrowheads. NB1 cell extracts were used as positive control for ALK expression in RH4 and RD immunoblots.

Mentions: To explore baseline expression and phosphorylation of ALK kinase, RMS cell lines representative of the ARMS and ERMS subtype, and previously defined for ALK expression [25], were utilized, together with neuroblastoma cells carrying amplified or mutant (F1174L) ALK gene. Consistent with our previous observations, ALK was expressed mainly in PAX3-FOXO1-positive RH30 cells, but lacked of constitutive kinase activity (Fig 1A). Similarly, basal phosphorylation of mutant ALK receptor was very low in SH-SY5Y cells compared to that of NB1 (amplified ALK), suggesting that under some circumstances protein expression overcomes gene mutational status as criteria for intrinsic kinase activity. However, to rule out aberrant protein trafficking and localization, cell surface detection of ALK was carried out using a monoclonal antibody directed toward the extracellular portion of the kinase, in cells kept in ice to avoid antigen modulation and internalization. When compared to ALK-amplified NB1 cells, PAX3-FOXO1-positive RH30 cells showed a weak but detectable ALK expression at the plasma membrane (Fig 1B), whereas in the other two RMS cell lines ALK signal was undistinguishable from background fluorescence. Indeed, when dimerization was induced by exposing cells to agonist mAb46 monoclonal antibody, activation of ALK occurred in RH30 cells, as indicated by the increased degree of phosphorylation of both the plasma membrane and intracellular pool of ALK (Fig 1C, lanes 6–7 and 8–9, respectively) [39,42]. Of note, antibody treatment acted on cell surface-exposed ALK receptor, since phosphorylation of both 220 and 140 kDa ALK (arrowheads) was observed, the latter of which corresponds to an extracellular product cleavage of the mature receptor by plasma membrane proteases [43]. These observations were confirmed in NB1 and SH-SY5Y neuroblastoma cells, which were responsive to antibody treatment independently of ALK mutational status (Fig 1, lane 8).


Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors.

Peron M, Lovisa F, Poli E, Basso G, Bonvini P - PLoS ONE (2015)

Endogenous ALK expression levels influence receptor intrinsic kinase activity in RMS cells.(A) ALK protein expression (upper panels) and phosphorylation (lower panel) in RMS (RH30, RH4, RD) and neuroblastoma (NB1, SH-SY5Y) cell lines. Western blot analysis of cell extracts using the specified polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved ALK form (140 kDa) are shown. ALK gene status is also indicated (A = amplified; M = mutated; WT = wild-type) (B) Cell surface detection and quantitative assessment of ALK receptor. Membrane-bound ALK kinase in non-permeabilized RMS and neuroblastoma cells using a primary monoclonal antibody against the N-terminal portion of ALK. Fluorescent-conjugated secondary antibody was used to acquire ALK signal with FACS-Calibur Cell Cytometer. ALK-amplified NB1 cells were included in the analysis as positive control for ALK expression and localization at the plasma membrane. (C) mAb46 effect on ALK receptor activation. RMS (RH30, RH4 and RD) and neuroblastoma (NB1, SHSY-5Y) cells were treated (+) or left untreated (-) with 1 μg/ml agonist mAb46 for 30 min. Membrane-bound (MB) and intracellular (IC) ALK was immunoprecipitated as described in material and methods and detected using polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved (140 kDa) ALK proteins position is indicated by arrowheads. NB1 cell extracts were used as positive control for ALK expression in RH4 and RD immunoblots.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493009&req=5

pone.0132330.g001: Endogenous ALK expression levels influence receptor intrinsic kinase activity in RMS cells.(A) ALK protein expression (upper panels) and phosphorylation (lower panel) in RMS (RH30, RH4, RD) and neuroblastoma (NB1, SH-SY5Y) cell lines. Western blot analysis of cell extracts using the specified polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved ALK form (140 kDa) are shown. ALK gene status is also indicated (A = amplified; M = mutated; WT = wild-type) (B) Cell surface detection and quantitative assessment of ALK receptor. Membrane-bound ALK kinase in non-permeabilized RMS and neuroblastoma cells using a primary monoclonal antibody against the N-terminal portion of ALK. Fluorescent-conjugated secondary antibody was used to acquire ALK signal with FACS-Calibur Cell Cytometer. ALK-amplified NB1 cells were included in the analysis as positive control for ALK expression and localization at the plasma membrane. (C) mAb46 effect on ALK receptor activation. RMS (RH30, RH4 and RD) and neuroblastoma (NB1, SHSY-5Y) cells were treated (+) or left untreated (-) with 1 μg/ml agonist mAb46 for 30 min. Membrane-bound (MB) and intracellular (IC) ALK was immunoprecipitated as described in material and methods and detected using polyclonal anti-ALK (ALK) and anti-phospho ALK (ALKY1604) antibodies. Full-length (220 kDa) and cleaved (140 kDa) ALK proteins position is indicated by arrowheads. NB1 cell extracts were used as positive control for ALK expression in RH4 and RD immunoblots.
Mentions: To explore baseline expression and phosphorylation of ALK kinase, RMS cell lines representative of the ARMS and ERMS subtype, and previously defined for ALK expression [25], were utilized, together with neuroblastoma cells carrying amplified or mutant (F1174L) ALK gene. Consistent with our previous observations, ALK was expressed mainly in PAX3-FOXO1-positive RH30 cells, but lacked of constitutive kinase activity (Fig 1A). Similarly, basal phosphorylation of mutant ALK receptor was very low in SH-SY5Y cells compared to that of NB1 (amplified ALK), suggesting that under some circumstances protein expression overcomes gene mutational status as criteria for intrinsic kinase activity. However, to rule out aberrant protein trafficking and localization, cell surface detection of ALK was carried out using a monoclonal antibody directed toward the extracellular portion of the kinase, in cells kept in ice to avoid antigen modulation and internalization. When compared to ALK-amplified NB1 cells, PAX3-FOXO1-positive RH30 cells showed a weak but detectable ALK expression at the plasma membrane (Fig 1B), whereas in the other two RMS cell lines ALK signal was undistinguishable from background fluorescence. Indeed, when dimerization was induced by exposing cells to agonist mAb46 monoclonal antibody, activation of ALK occurred in RH30 cells, as indicated by the increased degree of phosphorylation of both the plasma membrane and intracellular pool of ALK (Fig 1C, lanes 6–7 and 8–9, respectively) [39,42]. Of note, antibody treatment acted on cell surface-exposed ALK receptor, since phosphorylation of both 220 and 140 kDa ALK (arrowheads) was observed, the latter of which corresponds to an extracellular product cleavage of the mature receptor by plasma membrane proteases [43]. These observations were confirmed in NB1 and SH-SY5Y neuroblastoma cells, which were responsive to antibody treatment independently of ALK mutational status (Fig 1, lane 8).

Bottom Line: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation.We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity.However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity.

View Article: PubMed Central - PubMed

Affiliation: Clinica di Oncoematologia Pediatrica di Padova, Azienda Ospedaliera-Università di Padova, Padua, Italy.

ABSTRACT

Background: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation. This concept is essential from a therapeutic standpoint, as clinical evidence indicates that tumours carrying deregulated RTKs are particularly susceptible to their activity but also to their inhibition. Rhabdomyosarcoma (RMS) is an aggressive childhood cancer where emerging therapies rely on the use kinase inhibitors, and among druggable kinases ALK represents a potential therapeutic target to commit efforts against. However, the functional relevance of ALK in RMS is not known, likewise the multi-component deregulated RTK profile to which ALK belongs.

Methods: In this study we used RMS cell lines representative of the alveolar and embrional histotype and looked at ALK intracellular localization, activity and cell signalling.

Results: We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity. Indeed, increase of ALK phosphorylation was observed upon PTPase inhibition, as well as after ligand binding or protein overexpression. In these conditions, ALK signalling proceeded through the MAPK/ERK and PI3K/AKT pathways, and it was susceptible to ATP-competitive inhibitors exposure. However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity. Indeed, analysis of baseline and inducible RTK phosphorylation confirmed that RMS cells were susceptible to ALK kinase inhibitors even in the absence of the primary intended target, due to the presence of compensatory RTKs signalling pathways.

Conclusions: These data, hence, provided evidences of a potentially active role of ALK in RMS cells, but also suggest caution in considering ALK a major therapeutic target in this malignancy, particularly if expression and activity cannot be accurately determined.

No MeSH data available.


Related in: MedlinePlus